"The keeping is the problem": A qualitative study of IRB-member perspectives in Botswana on the collection, use, and storage of human biological samples for research.

BACKGROUND: Concurrent with efforts to establish national and regional biorepositories in Africa is widespread endorsement of ethics committees as stewards of the interests of individual donors and their communities. To date, ethics training programs for IRB members in Botswana have focused on ethical principles and international guidelines rather than on the ethical dimensions of specific medical technologies and research methodologies. Little is known about the knowledge and concerns of current and prospective IRB members in Botswana with respect to export, reuse, storage, and benefit-sharing of biospecimens. METHODS: This qualitative study examined perspectives of IRB members in Botswana about the collection and use of biospecimens in research. Forty-one IRB members representing five committees in Botswana participated in discussions groups in March 2013. Transcriptions of audiotapes and field notes were analyzed to identify issues of concern that might be alleviated through education and capacity-building, and areas that required ongoing discussion or additional regulatory guidance. RESULTS: Areas of concern included lack of understanding among patients and providers about the use of biospecimens in clinical care and research; reuse of biospecimens, particularly issues of consent, ownership and decision-making; export of specimens and loss of control over reuse and potential benefits; and felt need for regulatory guidance and IRB-member training. Local belief systems about bodily integrity and strong national identity in the construct of benefits may be at odds with initiatives that involve foreign biorepositories or consider such collections to be global public goods. CONCLUSION: Education is needed to strengthen IRB-member capacity to review and monitor protocols calling for the collection and use of biospecimens, guided by clear national policy on priority-setting, partnerships, review, and oversight. Engagement with local stakeholders is needed to harmonize fundamentally different ways of understanding the human body and community identity with the aims of contemporary biomedicine.

Clinical outcomes among persons with pulmonary tuberculosis caused by Mycobacterium tuberculosis isolates with phenotypic heterogeneity in results of drug-susceptibility tests.

BACKGROUND: Patients with multidrug-resistant (MDR) tuberculosis may have phenotypic heterogeneity in results of drug-susceptibility tests (DSTs). However, the impact of this on clinical outcomes among patients treated for MDR tuberculosis is unknown. METHODS: Phenotypic DST heterogeneity was defined as presence of at least 1 Mycobacterium tuberculosis isolate susceptible to rifampicin and isoniazid recovered <3 months after MDR tuberculosis treatment initiation from a patient with previous documented tuberculosis due to M. tuberculosis resistant to at least rifampicin and isoniazid. The primary outcome was defined as good (ie, cure or treatment completion) or poor (ie, treatment failure, treatment default, or death). A secondary outcome was time to culture conversion. Cox proportional hazard models were used to determine the association between phenotypic DST heterogeneity and outcomes. RESULTS: Phenotypic DST heterogeneity was identified in 33 of 475 patients (7%) with MDR tuberculosis. Poor outcome occurred in 126 patients (28%). Overall, patients with MDR tuberculosis who had phenotypic DST heterogeneity were at greater risk of poor outcome than those with MDR tuberculosis but no phenotypic DST heterogeneity (adjusted hazard ratio [aHR], 2.1; 95% confidence interval [CI], 1.2-3.6). Among HIV-infected patients with MDR tuberculosis, the adjusted hazard for a poor outcome for those with phenotypic DST heterogeneity was 2.4 (95% CI, 1.3-4.2) times that for those without phenotypic DST heterogeneity, whereas among HIV-negative patients with MDR tuberculosis, the adjusted hazard for those with phenotypic DST heterogeneity was 1.5 (95% CI, .5-4.3) times that for those without phenotypic DST heterogeneity. HIV-infected patients with MDR tuberculosis with phenotypic DST heterogeneity also had a longer time to culture conversion than with HIV-infected patients with MDR tuberculosis without phenotypic DST heterogeneity (aHR, 2.9; 95% CI, 1.4-6.0). CONCLUSIONS: Phenotypic DST heterogeneity among persons with HIV infection who are being treated for MDR tuberculosis is associated with poor outcomes and longer times to culture conversion.

Building research capacity through programme development and research implementation in resource-limited settings - the Ipabalele study protocol: observational cohort studies determining the effect of HIV on the natural history of cervical cancer in Botswana.

INTRODUCTION: The global burden of cancer continues to increase in low- and middle-income countries, particularly in sub-Saharan Africa (SSA). Botswana, a middle-income country in SSA, has the second highest prevalence of HIV worldwide and has seen an increase in human papillomavirus (HPV)-associated cervical cancer over the last decade in the setting of improved survival of HIV-infected women. There is an urgent need to understand more clearly the causes and consequences of HPV-associated cervical cancer in the setting of HIV infection. We initiated the Ipabalele ('take care of yourself' in Setswana) programme to address this need for new knowledge and to initiate long-term research programme capacity building in the region. In this manuscript, we describe the components of the programme, including three main research projects as well as a number of essential cores to support the activities of the programme. METHODS AND PROCEDURES: Our multidisciplinary approach aims to further current understanding of the problem by implementing three complementary studies aimed at identifying its molecular, behavioural and clinical determinants. Three participant cohorts were designed to represent the early, intermediate and late stages of the natural history of cervical cancer.The functional structure of the programme is coordinated through programmatic cores. These allow for integration of each of the studies within the cohorts while providing support for pilot studies led by local junior investigators. Each project of the Ipabalele programme includes a built-in capacity building component, promoting the establishment of long-lasting infrastructure for future research activities. ETHICS AND DISSEMINATION: Institutional review board approvals were granted by the University of Pennsylvania, University of Botswana and Ministry of Health and wellness of Botswana. Results will be disseminated via the participating institutions and with the help of the Community Advisory Committee, the project's Botswana advisory group.

Diagnosis of pulmonary tuberculosis and assessment of treatment response through analyses of volatile compound patterns in exhaled breath samples.

OBJECTIVES: We determined the performance of a sensor array (an electronic nose) made of 8 metalloporphyrins coated quartz microbalances sensors for the diagnosis and prognosis of pulmonary tuberculosis (TB) using exhaled breath samples. METHODS: TB cases and healthy controls were prospectively enrolled. Signals from volatile organic compounds (VOCs) in breath samples were measured at days 0, 2, 7, 14, and 30 of TB therapy and correlated with clinical and microbiological measurements. RESULTS: Fifty one pulmonary TB cases and 20 healthy HIV-uninfected controls were enrolled in the study. 31 (61%) of the 51 pulmonary TB cases were coinfected with HIV. At day 0 (before TB treatment initiation) the sensitivity of our device was estimated at 94.1% (95% confidence interval [CI], 83.8-98.8%) and specificity was 90.0% (95% CI, 68.3-98.8%) for distinguishing TB cases from controls. Time-dependent changes in the breath signals were identified as time on TB treatment progressed. Time-dependent signal changes were more pronounced among HIV-uninfected patients. CONCLUSION: The identification of VOCs' signals in breath samples using a sensor array achieved high sensitivity and specificity for the diagnosis of TB and allowed following signal changes during TB treatment.

Prostaglandins regulate transcription by means of prostaglandin response elements located in the promoters of mammalian Na,K-ATPase beta 1 subunit genes.

Prostaglandins are potent products of arachidonic acid metabolism that play significant roles in regulating ion transport in the kidney. In the Madin Darby canine kidney (MDCK) cell line prostaglandin E(1) (PGE(1)) stimulates the activity of the Na,K-ATPase and regulates transcription. Transient transfection studies conducted in MDCK cells with a human Na,K-ATPase beta1 subunit promoter/luciferase construct, pHbeta1-1141 Luc, showed a PGE(1) stimulation. The PGE(1) stimulation was inhibited by the PGE receptor antagonists SC19220 and AH6809, indicating the involvement of EP1 receptors (coupled to phospholipase C) and EP2 receptors (coupled to adenylate cyclase), respectively. A prostaglandin-regulatory element (PGRE) within the beta1 subunit promoter (-110 to -92, AGTCCCTGC) is sufficient to elicit a PGE(1) stimulation in a heterologous promoter (in pLUC-MCS). Studies with promoter mutants indicated that in addition to the PGRE, an adjacent Sp1 site was also essential for regulation by PGE(1). Consistent with the involvement of Sp1 are the results of DNA affinity precipitation studies, which indicate that Sp1 as well as CREB, and Sp3 all bind to the PGRE. The involvement of this PGRE in transcriptional regulation of the Na,K-ATPase beta1 gene was examined in a number of species. Only human and chimpanzee promoters possessed an identical PGRE site, unlike dog, rat, and mouse, which possessed Sp1 sites in similar locations. Two alternative PGREs were subsequently identified. The sequence of the one of these PGREs (TGACCTTC, -445 to -438) was conserved throughout all species examined, suggesting its physiologic significance.

Involvement of EP1 and EP2 receptors in the regulation of the Na,K-ATPase by prostaglandins in MDCK cells.

Prostaglandins are key regulators of ion transport in the kidney. In MDCK cells, which model distal tubule cells, the transcription of the Na,K-ATPase beta1 subunit is regulated by PGE1 and PGE2. To identify the EP receptors that mediate transcriptional regulation, transient transfection studies are conducted using the human beta1promoter/luciferase construct, pHbeta1-1141 Luc. The involvement of EP1 and EP2 receptors is indicated by studies with the EP1 selective agonist 17-phenyl trinor PGE2, and the EP2 selective agonist butaprost (which stimulate), as well as by studies with the antagonists SC-51089 (EP1 specific) and AH 6809 (EP1 and EP2 specific). Consistent with the involvement of Gs coupled EP2 receptors, is that the PGE1 stimulation is inhibited by the PKAI expression vector (encoding the protein kinase A (PKA) inhibitory protein), as well as by the myristolated PKA inhibitory peptide PKI. In addition to this evidence (for the involvement of EP2 receptors), evidence for the involvement of EP1 receptors in the PGE1 mediated stimulation of Na,K-ATPase beta subunit gene transcription includes the stimulatory effect of 17-phenyl trinor PGE2, as well as the inhibitory effects of SC-51089. Also consistent with the involvement of Gq coupled EP1 receptors, the PGE1 stimulation is inhibited by the PKCI vector (encoding the PKC inhibitory domain), the PKC inhibitor Go 6976, thapsigargin, as well as the calmodulin antagonists W7 and W13.

Regulation of the Na-K-ATPase beta(1)-subunit promoter by multiple prostaglandin-responsive elements.

Renal prostaglandins modulate the activity of a number of the transport systems in the kidney, including the Na-K-ATPase. Not only do prostaglandins have acute affects on renal Na-K-ATPase, but in addition prostaglandins have chronic affects, which include regulation at the transcriptional level. Previously, we have presented evidence that one such prostaglandin, PGE(1), stimulates the transcription of the human Na-K-ATPase beta(1)-subunit gene in Madin-Darby canine kidney cells via cAMP- and Ca(2+)-mediated pathways (Taub M, Borsick M, Geisel J, Matlhagela K, Rajkhowa T, and Allen C. Exp Cell Res 299: 1-14, 2004; Matlhagela K, Borsick M, Rajkhowa T, and Taub M. J Biol Chem 280: 334-346, 2005). Evidence was presented indicating that PGE(1) stimulation was mediated through the binding of cAMP-regulatory element binding protein (CREB) to a prostaglandin-responsive element (PGRE) as well as Sp1 binding to an adjacent Sp1 site. In this report, we present evidence from EMSAs and DNA affinity precipitation studies that another PGRE present in the Na-K-ATPase beta(1)-subunit promoter similarly binds CREB and Sp1. The evidence that indicates a requirement for CREB as well as Sp1 for gene activation through both PGREs (PGRE1 and PGRE3) includes studies with a dominant negative CREB (KCREB), Drosophila SL2 cells, and PGRE mutants. The results of these studies are indicative of a synergism between Sp1 and CREB in mediating regulation by PGRE3; while regulation occurring through PGRE1 also involves Sp1 and CREB, the mechanism appears to be distinct.

Identification of a prostaglandin-responsive element in the Na,K-ATPase beta 1 promoter that is regulated by cAMP and Ca2+. Evidence for an interactive role of cAMP regulatory element-binding protein and Sp1.

The Na,K-ATPase is a transmembrane protein responsible for maintaining electrochemical gradients across the plasma membrane in all mammalian cells, a process that is subject to regulation at the transcriptional as well as post-transcriptional level. Included among physiologic regulators in the kidney are prostaglandins. Previously, we demonstrated that prostaglandin E(1) (PGE(1)) increases the activity and expression of the Na,K-ATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and Allen, C. (2004) Exp. Cell Res. 299, 1-14; Taub, M. L., Wang, Y., Yang, I. S., Fiorella, P., and Lee, S. M. (1992) J. Cell. Physiol. 151, 337-346). In this work, we present evidence that transcription of the Na,K-ATPase beta(1) subunit is stimulated by PGE(1), an effect that may be mediated through the cAMP and Ca(2+) pathways. Transient transfection studies using 5'-deletion mutants of the human beta(1) subunit promoter indicated that region -100 to -92 containing the sequence AGTCCCTGC (a prostaglandin-responsive element (PGRE)) is required to elicit the stimulatory effects of PGE(1), 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and okadaic acid. Electrophoretic mobility shift assays indicated that both the cAMP regulatory element-binding protein (CREB) and Sp1 bind to the PGRE present within this region of the beta(1) subunit promoter. The involvement of the PGRE and Sp1 sites in regulation by PGE(1) was further confirmed by the increased PGE(1) stimulation that was observed following insertion of the PGRE into a promoter/luciferase construct containing a portion of a heterologous promoter and the fibronectin promoter with four GC boxes. Further evidence suggesting an interaction between Sp1 and CREB was obtained from experiments conducted with pLuc-MCS-beta 72-167, which contains region -167 to -72 in the human beta(1) subunit promoter. The PGE(1) stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-beta 72-167 was reduced when the two GC boxes immediately upstream from the PGRE were translocated farther upstream. Also consistent with an interaction between CREB and Sp1 are the results of our immunoprecipitation studies indicating that CREB co-immunoprecipitated with Sp1 when an antibody against CREB, Sp1, or the CREB-binding protein was used.

Regulation of the Na,K-ATPase in MDCK cells by prostaglandin E1: a role for calcium as well as cAMP.

Prostaglandins (PGs) play a significant role in the regulation of sodium reabsorption by the kidney, in addition to accumulating during inflammation as well as in several solid tumors. Previously, we presented evidence indicating that prostaglandin E(1) (PGE(1)), a supplement in the serum-free medium for MDCK cells, increases the activity of the Na,K-ATPase in MDCK cells, in addition to its growth stimulatory effect [J. Cell. Physiol. 151 (1992) 337]. This report defines the molecular mechanisms, and signaling pathways responsible for the increased Na,K-ATPase activity. Our results indicate that the increased activity of the Na,K-ATPase in MDCK monolayers treated with either PGE(1) or 8Bromocyclic AMP (8Br-cAMP) can be attributed to an increase in the rate of biosynthesis of the Na,K-ATPase, and an increase in the levels of Na,K-ATPase alpha and beta subunit mRNAs. As beta subunit mRNA increased to a larger extent than alpha subunit mRNA, transient transfection studies were conducted using a human beta1 promoter/luciferase construct [Nucleic Acids Res. 21 (1993) 2619]. While an 8Br-cAMP stimulation was observed (suggesting the involvement of cAMP), our results also suggest that the observed PGE(1) stimulation could be explained by the involvement of Ca(2+) as well protein kinase C (PKC). Consistent with the involvement of Ca(2+), TMB-8 (which inhibits Ca(2+) efflux from intracellular stores) inhibited the PGE(1) stimulation. Moreover, PGE(1) was observed to stimulate the translocation of PKC beta1 from the soluble to the particulate fraction. The translocation of PKC, the PGE(1) stimulation of transcription, and the PGE(1)-mediated increase in the beta subunit mRNA level were all inhibited by the PKC inhibitor Gö6989. These results can be explained by the involvement of two classes of cell surface receptors in mediating the PGE(1) stimulation, including the EP1subtype (which activates phospholipase C), as well as the EP2 subtype (which activates adenylate cyclase).